Project name: Halobacterium salinarum NRC-1

Description: Cells responding to dramatic environmental changes or undergoing a developmental switch typically change the expression of numerous genes. In bacteria sigma factors regulate much of this process while in eukaryotes four RNA polymerases and a multiplicity of generalized transcription factors (GTFs) are required. Here, using a systems approach, we provide experimental evidence (including protein-coIP, ChIP-Chip, GTF perturbation and knockout, and measurement of transcriptional changes in these genetically perturbed strains) for how archaea likely accomplish similar large-scale transcriptional segregation and modulation of physiological functions. We are able to associate GTFs to nearly half of all putative promoters and show evidence for at least 7 of the possible 42 functional GTF pairs. This report represents a significant contribution towards closing the gap in our understanding of gene regulation by GTFs for all three domains of life and provides an example for how to utilize various experimental techniques to rapidly learn significant portions of a global gene regulatory network of organisms for which little is previously known.Keywords: ChIP-ChipOverall design: Halobacterium NRC-1 expressing Cmyc-tagged TFBs and TBPs were grown to an OD600 between 1 and 1.2. The cells were cross-linked with 1.2% formaldehyde, then lysed and DNA sheared via sonication. Next cleared cell lysate was subject to immunoprecipitation against an anti-myc antibody. Protein-DNA complexes were purified and DNA was isolated after protease and RNAase treatment. DNA was amplified using universal linkers, labeled directly using Kreatech 547 and 647 dyes. Samples were grown in biological duplicate. Each DNA sample was hybridized against 2 microarrays as dyefilps. At least two experiments (including biological duplicates) were carried out.

Data type: Epigenomics

Sample scope: Multiisolate

Relevance: Environmental

Release date: 2007-02-23

Last updated: 2007-02-15